CYP3A deficiency alters bile acid homeostasis and leads to changes in hepatic susceptibility in rats.

Cytochrome P450 3A (CYP3A) as an important enzyme metabolizes many drugs and a variety of endogenous substances. Bile acids (BA) regulate physiological function by activating BA receptors. In this study, CYP3A1/2 gene knockout (KO) and wild-type (WT) rats were used to investigate the regulatory effects of CYP3A on BA homeostasis and liver function. Compared with WT rats, BA concentrations in serum, liver and small intestine of CYP3A1/2 KO rats increased significantly, which was due to the decrease of catabolism and the increase of synthesis. In particular, the composition of serum BA (overall hydrophobicity) presented an age- and CYP3A-dependent manner. With the aging of WT rats, the serum BA became more hydrophobic, while this trend was delayed in CYP3A1/2 KO rats. Moreover, the level of serum total cholesterol, the precursor of BA synthesis, decreased by about 20% in CYP3A1/2 KO rats, which is due to the low synthesis but high biotransformation rate. The increase of BA pool further led to the change of transcription level of BA receptor in liver (pregnane X receptor) and small intestine (Takeda G-protein receptor 5), and affected the function and morphology of CYP3A1/2 KO rat liver. In conclusion, CYP3A is a key regulator of BA homeostasis in rats, especially in regulating BA pool size, composition and balance of anabolism, and prevents susceptibility to hepatotoxicity under BA overload.

Heme Binding Biguanides Target Cytochrome P450-Dependent Cancer Cell Mitochondria.

The mechanisms by which cancer cell-intrinsic CYP monooxygenases promote tumor progression are largely unknown. CYP3A4 was unexpectedly associated with breast cancer mitochondria and synthesized arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promoted the electron transport chain/respiration and inhibited AMPKα. CYP3A4 knockdown activated AMPKα, promoted autophagy, and prevented mammary tumor formation. The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active-site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 AA epoxygenase activity. HBB also inhibited growth of established ER+ mammary tumors and suppressed intratumoral mTOR. CYP3A4 AA epoxygenase inhibition by biguanides thus demonstrates convergence between eicosanoid activity in mitochondria and biguanide action in cancer, opening a new avenue for cancer drug discovery.

CRISPR knockout rat cytochrome P450 3A1/2 model for advancing drug metabolism and pharmacokinetics research.

Cytochrome P450 (CYP) 3A accounts for nearly 30% of the total CYP enzymes in the human liver and participates in the metabolism of over 50% of clinical drugs. Moreover, CYP3A plays an important role in chemical metabolism, toxicity, and carcinogenicity. New animal models are needed to investigate CYP3A functions, especially for drug metabolism. In this report, Cyp3a1/2 double knockout (KO) rats were generated by CRISPR-Cas9 technology, and then were characterized for viability and physiological status. The Cyp3a1/2 double KO rats were viable and fertile, and had no obvious physiological abnormities. Compared with the wild-type (WT) rat, Cyp3a1/2 expression was completely absent in the liver of the KO rat. In vitro and in vivo metabolic studies of the CYP3A1/2 substrates indicated that CYP3A1/2 was functionally inactive in double KO rats. The Cyp3a1/2 double KO rat model was successfully generated and characterized. The Cyp3a1/2 KO rats are a novel rodent animal model that will be a powerful tool for the study of the physiological and pharmacological roles of CYP3A, especially in drug and chemical metabolism in vivo.

Metabolomic screening and identification of the bioactivation pathways of ritonavir.

Ritonavir-boosted protease inhibitor regimens are widely used for HIV chemotherapy. However, ritonavir causes multiple side effects, and the mechanisms are not fully understood. The current study was designed to explore the metabolic pathways of ritonavir that may be related to its toxicity. Metabolomic analysis screened out 26 ritonavir metabolites in mice, and half of them are novel. These novel ritonavir metabolites include two glycine conjugated, two N-acetylcysteine conjugated, and three ring-open products. Accompanied with the generation of ritonavir ring-open metabolites, the formation of methanethioamide and 2-methylpropanethioamide were expected. Upon the basis of the structures of these novel metabolites, five bioactivation pathways are proposed, which may be associated with sulfation and epoxidation. By using Cyp3a-null mice, we confirmed that CYP3A is involved in four pathways of RTV bioactivation. In addition, all these five bioactivation pathways were recapitulated in the incubation of ritonavir in human liver microsomes. Further studies are suggested to determine the role of CYP3A and these bioactivation pathways in ritonavir toxicity.

Absence of both cytochrome P450 3A and P-glycoprotein dramatically increases docetaxel oral bioavailability and risk of intestinal toxicity.

Docetaxel is one of the most widely used anticancer drugs. A major problem with docetaxel treatment, however, is the considerable interpatient variability in docetaxel exposure. Another disadvantage of the drug is that it has a very low oral bioavailability and can therefore only be administered i.v. The drug-metabolizing enzyme cytochrome P450 3A (CYP3A) and the drug transporter P-glycoprotein (P-gp; MDR1) are considered to be major determinants of docetaxel pharmacokinetics. It has been hypothesized that CYP3A and P-gp work synergistically in limiting the systemic exposure to many orally ingested drugs. However, it has been difficult to examine this interplay in vivo. We therefore generated mice lacking all CYP3A and P-gp genes. Although missing two primary detoxification systems, Cyp3a/Mdr1a/1b(-/-) mice are viable, fertile, and without spontaneous abnormalities. When orally challenged with docetaxel, a disproportionate (>70-fold) increase in systemic exposure was observed compared with the increases in single Cyp3a(-/-) (12-fold) or Mdr1a/1b(-/-) (3-fold) mice. Unexpectedly, although CYP3A and P-gp collaborated extremely efficiently in lowering docetaxel exposure, their individual efficacy was not dependent on activity of the other protein. On reflection, this absence of functional synergism makes biological sense, as synergism would conflict with a robust detoxification defense. Importantly, the disproportionate increase in docetaxel exposure in Cyp3a/Mdr1a/1b(-/-) mice resulted in dramatically altered and lethal toxicity, with severe intestinal lesions as a major cause of death. Simultaneous inhibition of CYP3A/P-gp might thus be a highly effective strategy to improve oral drug bioavailability but with serious risks when applied to drugs with narrow therapeutic windows.

Defective activity of recombinant cytochromes P450 3A4.2 and 3A4.16 in oxidation of midazolam, nifedipine, and testosterone.

Cytochrome P4503A4 (CYP3A4) is the most abundant cytochrome P450 in adult human liver and small intestine and oxidizes numerous clinically, physiologically, and toxicologically important compounds. The metabolic activity of CYP3A4 in patients varies at least 10-fold in vivo, and CYP3A4 genetic variants are considered one of the causes of individual differences. The cDNAs for the CYP3A4(*)2 (S222P), (*)7 (G56D), (*)16 (T185S), and (*)18 (L293P) mutant alleles, found in high frequencies in Caucasians or Asians, were constructed by site-directed mutagenesis and expressed in an Escherichia coli expression system. Midazolam (MDZ), testosterone (TST), and nifedipine (NIF) were used to assess the catalytic activities of the CYP3A4 wild type (CYP3A4.1) and its variants. The catalytic activities of CYP3A4.2 and CYP3A4.16 were reduced (lower V(max) and increased K(m) relative to CYP3A4.1) for all substrates. The CYP3A4.7 showed lower V(max) values for MDZ and NIF (60 and 84%, respectively) and a higher K(m) (2-fold) for TST but not for MDZ or NIF. Although CYP3A4.18 showed low V(max) values for MDZ, NIF, and TST (88, 72, and 80% of CYP3A4.1, respectively), no significant differences were identified in the ratio V(max)(/K)(m). In summary, CYP3A4.2 and CYP3A4.16 exhibited significantly lower activity for MDZ, TST, and NIF oxidations than CYP3A4.1. Therefore, drugs metabolized by only CYP3A should be carefully administered to patients with these alleles.